Cellulose is the major structural component of higher plants and occurs naturally in almost pure form only in cotton fiber. It provides plant cells with high tensile strength helping them to resist mechanical stress and osmotic pressure. Cellulose is a linear polysaccharide of glucose residues connected by β-1,4-linkages. In nature cellulose is usually associated with lignin and hemicelluloses. Cellulosic material is degraded in nature by a number of various organisms including bacteria and fungi. The biological conversion of cellulose to glucose generally requires three major groups of enzymes: cellobiohydrolases (CBH), endoglucanases (EG) and beta-glucosidases (BG).
There is a wide spectrum of industrial applications of cellulases. In the textile industry, cellulases are used in denim finishing to create a fashionable stone washed appearance in denim cloths in a biostoning process. Cellulases are also used, for instance, to clean fuzz and prevent formation of pills on the surface of cotton garments. In detergent industry cellulases are used to brighten colors and to prevent graying and pilling of garments. Cellulases are further used in food industry and animal feed manufacturing, and they have a great potential in the pulp and paper industry, for instance, in deinking to release ink from fiber surfaces and in improving pulp drainage.
Industrially used cellulases are often mixtures of enzymes having a variety of activities and substrate specificities. The commercial enzyme preparations often comprise all three cellulase activities i.e. CBH, EG and BG. In addition the unique properties of each cellulase make some more suitable for certain purposes than others, and therefore efforts have also been made to obtain and use cellulases having only the desired activities. The most widely used cellulases of fungal origin are derived from Trichoderma reesei. However, also other fungal sources have been suggested e.g., in U.S. Pat. No. 5,457,046.
Cellulases applied in denim treatment are usually divided into two main groups: acid and neutral cellulases. Acid cellulases typically operate at pH 4.0 to 5.5 and neutral cellulases in the range of pH 6 to 8. Cellulases having characteristics of both the acid and neutral group can be called hybrid cellulases. Acid cellulases used in biostoning mainly originate from Trichoderma reesei (sexual form Hypocrea jecorina) and the neutral cellulases come from a variety of fungi, including genera of Melanocarpus, Humicola, Myceliophthora, Fusarium, Acremonium, and Chrysosporium (Haakana et al. 2004). T. reesei enzymes include, e.g., cellulases from the glycosyl hydrolase family 5 (endoglucanase II, EGII), family 7 (cellobiohydrolase 1, CBHI) and family 12 (endoglucanase III, EGIII; Ward et al. 1993), and the neutral cellulases, most often endoglucanases, from family 45 and family 7 (Henrissat, 1991; Henrissat and Bairoch, 1993, 1996).
U.S. Pat. No. 5,874,293 discloses an improved cellulase composition comprising elevated amounts of EGII endoglucanase of T. reesei for treating cellulose-containing textiles. The composition improved color properties, increased lightness and visual appearance and reduced pilling tendencies. WO97/14804 discloses one 20 kDa and one 50 kDa cellulase with endoglucanase activity derived from Melanocarpus sp. that are especially useful in the textile and detergent industry. Fusion proteins containing the 20K- or 50K-proteins linked to a cellulose binding domain preferably from Trichoderma reesei are suggested for creating new enzyme properties. No specific examples are given, nor are the desired properties described.
However, there is still a need for improved cellulases, including endoglucanases that are more efficient in fabric treatment and in other fields, where cellulases traditionally are used. In particular, there is a continuous need for more efficient cellulases to improve the process economics. The present invention aims to meet these needs.
In textile industry a “stone washed” look or an abraded look has been denim producers' interest in recent years. Traditional stone washing with pumice stones reduces the strength of fabric and burdens the laundering apparatuses. There is a trend towards enzymatic denim finishing processes, and cellulases have replaced or are being used together with pumice stones to give the fabric its desired “worn” look. Controlled enzyme treatments result in less damage to the garments and machines and eliminate the need for disposal of stones.
A general problem associated with enzymatic stone washing is backstaining caused by redeposition of removed Indigo dye during or after abrasion. The redepositioning of Indigo dye reduces the desired contrast between the white and indigo dyed yarns and it can be most easily noted on the reverse side of denim and the interior pockets (as increased blueness). On the face side this may be seen as reduced contrast between dyed areas and areas from which dye has been removed during biostoning. Backstaining can be reduced by using antibackstaining agents such as nonionic ethoxylated alcohols during the treatment or adding bleaching agents during rinsing steps. The nature of the enzyme has an impact on backstaining. Generally neutral cellulases backstain less than acid cellulases.
WO97/09410 describes that the addition of a certain type of cellulase to another cellulase having abrading activity reduces backstaining. The additional cellulase belongs to family 5 or 7, but it has no significant abrading effect in itself. Preferably said additional cellulase originates from Bacillus or Clostridium. 
U.S. Pat. No. 5,916,799 discloses cellulase compositions containing both cellobiohydrolases and endoglucanases that have been subjected to limited proteolysis to separate the core and binding domains of the enzymes. The obtained enzyme compositions were found to reduce backstaining. WO 94/07983 relates to the finding that redeposition of colorant onto fabric during the stone washing process can be reduced by employing a fungal cellulase composition, which is substantially free of cellobiohydrolase type components. WO96/23928 discloses treatment of cellulose containing fabrics with truncated cellulase enzymes. The truncated enzymes lacking cellulose binding domain (CBD) were found to reduce redeposition of dye and increase abrasion.
The general conclusion of the three references cited above could be that the cellulose binding domain has a negative effect on backstaining. However, the present invention now provides a cellulase construction with low backstaining property, despite the presence of a cellulose binding domain.